AU2016315795C1 - Fluorescence histo-tomography (FHT) systems and methods - Google Patents

Abstract

In one embodiment, a fluorescence histo-tomography (FHT) system is disclosed. The FHT system includes a housing, a fluorescence camera located within the housing, a white light camera located within the housing, and a fluorescence light source located within the housing. The FHT system further includes a support mount configured to support the housing within a chamber of a slicing apparatus such that the cameras and fluorescence light source are aimed towards a block face of a tissue specimen retained within the chamber.

Description

FLUORESCENCE HISTO-TOMOGRAPHY (FHT) SYSTEMS AND METHODS RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/211,930, filed on August 31, 2015, entitled "CRYOFLUORESCENCE TOMOGRPAHY (FHT) SYSTEMS AND METHODS," by Hoppin, et al., and to U.S. Non-Provisional Application No. 15/158,928, filed on May 19,2016, entitled "MULTI-SPECTRAL THREE DIMENSIONAL IMAGING SYSTEM AND METHOD," which claims priority to U.S. Provisional Application No. 62/164,800, filed on May 21, 2015, entitled "MULTI-SPECTRAL THREE DIMENSIONAL IMAGING SYSTEM AND METHOD," the contents all of which are herein incorporated by reference.

TECHNICAL FIELD

The present disclosure relates generally to imaging and, more specifically, to

fluorescence histo-tomography (FHT) systems and methods.

BACKGROUND

Multispectral fluorescence tissue slice imaging can be used to measure drug distribution

ex-vivo in standalone or retrofitted slice imagers. Such specificity in a single imaging system can

result in a high cost per scan. The inventors have identified that, for high throughput and low cost,

it would be valuable to construct a fluorescence histological (histo-) imager with a corresponding

software package that can work in tandem with slicing instruments. To that end, the methods

outlined here demonstrate a workflow of fluorescence imaging techniques for a versatile,

transportable add-on to existing histological slicing instruments.

It is desired to overcome or alleviate one or more difficulties of the prior art, or to at least

provide a useful alternative.

SUMMARY

In accordance with some embodiments of the present invention, there is provided a fluorescence histo-tomography (FHT) system comprising: a housing having a handle; one or more cameras located within the housing; a fluorescence excitation light source located within the housing; and a support mount coupled to a slicing apparatus and configured to removably position the housing with respect to a chamber of the slicing apparatus such that the one or more cameras and fluorescence excitation light source are aimed towards a block face of a tissue specimen retained within the chamber.

In accordance with some embodiments of the present invention, there is provided a method of performing fluorescence histo-tomography (FHT) comprising: capturing, by an imaging device mounted with respect to a chamber of a slicing apparatus by a support mount coupled to the slicing apparatus and configured to removably position the imaging device with respect to the chamber, a first image of a first block face of a tissue specimen retained within the chamber; capturing, by the imaging device, a second image of the first block face under fluorescence excitation illumination; and processing at least the first image and the second image to form a combined image.

According to one or more embodiments of the disclosure as described in greater detail

) below, a fluorescence histo-tomography (FHT) system is disclosed. The FHT system includes a

housing, a fluorescence camera located within the housing, a white light camera located within

the housing, and a fluorescence light source located within the housing. The FHT system further

5 includes a support mount configured to support the housing within a chamber of a slicing

apparatus such that the cameras and fluorescence light source are aimed towards a block face of

a tissue specimen retained within the chamber.

In further embodiments, a method for performing FHT is disclosed. The method includes

capturing, by an imaging device mounted within a chamber of a slicing apparatus, a white light

0 image of a block face of a tissue specimen retained within the chamber. The method also

includes capturing, by the imaging device, a fluorescence image of the block face under white

light and fluorescence illumination. The method further includes co-registering, by the imaging

device, the white light and fluorescence images to form a combined image. The method

additionally includes providing, by the imaging device, the combined image to an electronic

5 display.

In additional embodiments, a FHT system is disclosed. The FHT system includes means

for imaging a block face of a tissue specimen retained within a chamber of a slicing apparatus.

The FHT system also includes means for supporting the imaging means within the chamber of

the slicing apparatus.

2A

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of

this patent or patent applicationpublication with color drawing(s) will be provided by the Office

upon request and payment of the necessary fee.

The foregoing and other objects, features, aspects and advantages of the embodiments

disclosed herein will become more apparent from the following detailed description when taken

in conjunction with the following accompanying drawings.

FIGS. 1A-1B illustrate an example cryostat/cryomicrotome, according to various

embodiments.

FIGS. 2-4 illustrate an example fluorescence histo-tomography (FHT) imaging device for

use in a microtome or cryomicrotome, according to various embodiments.

FIG. 5 illustrates an example computing device of a FHT system, according to various

embodiments.

FIG. 6 illustrates an example simplified procedure for preparing a tissue specimen for

FHT imaging, according to various embodiments.

FIG. 7 illustrates an example simplified procedure for imaging a block face of a tissue

specimen using FHT imaging, according to various embodiments.

FIG. 8 illustrates an example registration scheme for images captured by a FHT system,

according to various embodiments.

FIGS. 9A-91 illustrate test results of FHT imaging of a fluorophore-infused piece of

twine, according to various embodiments.

FIGS. 10A-10B illustrate test results of FHT imaging of brain tissue, according to various

embodiments.

FIGS. 11A-11F illustrate further test results of FHT imaging of brain tissue, according to

various embodiments.

FIGS. 12A-12I illustrate additional examples of FHT imaging of brain tissue, according

to various embodiments.

FIG. 13A-13E illustrate examples of the imaging of intrathecally-administered anti-sense

oligonucleotides, according to various embodiments.

FIGS. 14A-14C illustrate examples of a mechanism to remove stuck tissues slices from

an FHT system.

FIGS. 15A-15B illustrate an example apparatus for precisely placing fiducial markers in

a tissue block.

FIG. 16 illustrates an example of multi-colored fiducial markers.

It should be understood that the above-referenced drawings are not necessarily to scale,

presenting a somewhat simplified representation of various preferred features illustrative of the

basic principles of the disclosure. The specific design features of the present disclosure,

including, for example, specific dimensions, orientations, locations, and shapes, will be

determined in part by the particular intended application and use environment.

DETAILED DESCRIPTION

Various challenges persist to find otherwise invisible, small objects in a body. Labeling

such objects with fluorophores is a possible technique to introduce contrast, but visible

fluorophores are problematic because excitation light and emission light are both absorbed and

scattered. Near-infrared (NIR) fluorescent light has the ability to provide high contrast in the

context of normal bodily tissues and fluids.

Histological analysis of sections from previously living tissue is time-consuming and

laborious because each section needs to be mounted, processed, and scanned (e.g., using a

microscope). Block face imaging is the opposite of conventional analysis because the tissue

slice is thrown away and only the exposed block face of the tissue specimen is imaged. When

using NIR fluorescent light to image the block face, extremely high signal to noise is achieved,

although one also has to attempt to compensate for the "extra depth" that NIR can see. In some

implementations, video information (e.g., color, grayscale, etc.) as well as assumptions about

light propagation in the tissue can be used to generate at least an approximation of the actual

fluorescence.

In some aspects, the techniques described herein utilize simultaneous color/NIR

acquisition to acquire data sets and mathematical methods applied to these data sets to

reconstruct in ultra-high resolution, at least an approximation of the actual NIR fluorescence

present in the tissue block and therefore the original living tissue. NIR also provides high

sensitivity and less interference from endogenous chromophores that would otherwise preclude

visible fluorescence imaging.

Referring now to FIG. 1A, a cryostat/cryomicrotome 100 is shown, according to various

embodiments. In general, a microtome is a device typically used to prepare histologic samples

of a tissue block for use in microscopy. For example, a microtome may shave a thin layer of a

tissue specimen from a tissue block that can then be mounted to a slide for observation using a

microscope. A cryomicrotome is a specialized form of microtome operable to keep the tissue

specimen at a reduced temperature during slicing. While the FHT system disclosed herein is

described with respect to a cryomicrotome, the teachings herein are not limited as such. In

particular, the FHT system can be implemented using any number of different slicing devices

(e.g., other microtomes, histological sample preparation devices, macrotomes, etc.).

As shown, cryomicrotome 100 may include a housing 102 that encompasses a sealed

chamber 106 in which the cutting operation is performed. Cryomicrotome 100 may also include

a door 104 or other access mechanism (e.g., lid, etc.) that afford the user of cryomicrotome 100

access to chamber 106. During use, cryomicrotome 100 may regulate the internal temperature of

chamber 106 to prevent a frozen tissue specimen being sliced from thawing back to room

temperature.

FIG. 1B illustrates an example chamber 106 of cryomicrotome 100 in greater detail. As

shown, cryomicrotome 100 may include a specimen retainer 110 that holds a frozen specimen

112 in place. In addition, cryomicrotome 100 may also include one or more cutting blades 108

that, when actuated, moves across the block face of specimen 112 to remove a thin layer of

specimen 112. In many cases, specimen 112 may be any form of biological material extricated

from a subject and may be frozen in an optimal cutting temperature (OCT) material, prior to

placement in cryomicrotome 100.

The alignment of cutting blade 108 and specimen retainer 110 may be configurable in

some cryomicrotomes to adjust, e.g., the thickness of the resulting tissue slice and/or to ensure

proper contact of cutting blade 108 with tissue specimen 112. For example, a user of

cryomicrotome 100 may first ensure the correct positions of tissue specimen 112 before initiating

slicing of the tissue specimen 112. In some embodiments, actuation of cutting blade 108 may be

performed manually by the user. In other embodiments, actuation of cutting blade 108 and/or

movement of specimen retainer 110 may be performed automatically by cryomicrotome 100

under computerized control.

An exemplary cryomicrotome that can be used to implement the techniques herein is the

Leica 3050 cryomicrotome available from Leica Camera AG, Wetzlar, Germany. However, as

would be appreciated, the techniques herein can be applied to any number of different

cryomicrotomes and are not limited to a particular make, model, or type of cryomicrotome.

As noted above, NIR fluorescence imaging has emerged in recent years and demonstrates

the ability to produce images with high contrast between fluorophore infused tissue and regular

tissue. Table 1 below provides a listing of exemplary fluorophore agents with their peak

excitation wavelengths (Ex) and peak emission wavelengths (Em).

Type Agent Ex (nm) Em (nm) Reactive and Rated es Hydroxycoumarin 325 386 conjugated probes Aminocoumarin 350 455 Methoxycoumarin 360 410 Cascade Blue 375; 400 423 Lucifer Yellow 425 528 NBD 466 539 R-Phycoerythrin (PE) 480; 565 578 PE-Cy5 conjugates 480; 565; 650 670

PE-Cy7 conjugates 480:565;743 767 APC-Cy7 conjugates 650; 755 767 Red 613 480; 565 613 Fluorescein 495 519 FluorX 494 520 BODIPY-FL 503 512 TRITC 547 574 X-Rhodamine 570 576 Lissanine Rhodamine B5710 590 B PerCP 490 675 Texas Red 589 615 Allophycocyanin 650 660 (APC) TruRed 490,675 695 Alexa Fluor 350 346 445 Alexa Fluor 430 430 545 Alexa Fluor 488 494 517 Alexa Fluor 532 530 555 Alexa Fluor 546 556 573 Alexa Fluor 555 556 573 Alexa Fluor 568 578 603 Alexa Fluor 594 590 617 Alexa Fluor 633 621 639 Alexa Fluor 647 650 688 Alexa Fluor 660 663 690 Alexa Fluor 680 679 702 Alexa Fluor 700 696 719 Alexa Fluor 750 752 779 (y2 489 506 Cy3 (512); 550 570;(615) Cy3,5 581 596; (640) Cy5 (625); 650 670 Cy5,5 675 694 Cy7 743 767 ZW800-1 760 790 ZW700-1 Forte 675 700 Lipo8OO-Forte 760 790

Lipo700-Forte 675 700 Nucleic acid probes Hoeschst 33342 343) 483 DAPII 345 4554 Hlochlst 33258 345 478 SYTOX Blue 431 480 Chromomycin A3 445 575 Mithrarnvcin 445 575 YOYO-1I 491 509 syTrOXGreen 504 523 SYTOX Orange 547 570 Ethidium Borinide 493 620 7-AALD 546 647 Acridine Orange 503 530/640 TOO1,TOPR- 509 533 Thiazole Orange 510) 530 PropidiurnIodide (PI) 536 617 TOTO-3,TO-PRO-3 642 661 LDS 751 543; 590 712; 607 Fluorescent Proteins Y66F 360) 508 Y66[-1 360) 4.2 EBFP 380 440 Wild-type 396,475 5,,503 GFPuv 38 5 508 ECFP 434 477 Y66W 436 485 S65A 4-71 504 S65C 479 507 S65L 484 510 S65T 488 511 EGFP 489 508 EYFp 514 527! DsRed 558 583 Other probes Monochlorobirnane 380 461 _____________Caicein 496 517 Table 1

In many cases, the fluorophores that can be used for purposes of fluorescence imaging

may exhibit emission spectra in the range of approximately 200 nm to 1000 nm. Further, a

plurality of fluorophores can be used on a single specimen if each of the used fluorophores has

different emission spectra. Other fluorophores not listed in Table 1 can also be used without

deviating from the teachings herein.

According to various embodiments, NIR fluorescence imaging can be leveraged with a

cryomicrotome to perform fluorescence histo-tomography (FHT) of a tissue specimen. As noted,

cryomicrotomes are typically used to prepare tissue slices for further analysis (e.g., by mounting

a slice to a microscope slide, etc.). In contrast, the FHT techniques herein propose the exact

opposite, i.e., by imaging the exposed block face of the tissue specimen itself while in the

cryomicrotome without any regard to the resultant tissue slices, which may be discarded or

retained as desired by the user.

Referring now to FIGS. 2-4, a fluorescence histo-tomography (FHT) system is shown, in

various embodiments. As shown in FIG. 2, the FHT system may include an imaging

device/system 200 that is operable to image the exposed block face of tissue specimen 112

within chamber 106 of cryomicrotome 100. As would be appreciated, imaging system 200 may

be transportable, allowing imaging system 200 to be used with any number of different types of

cryomicrotomes. Advantageously, this allows a user to adapt a cryomicrotome to perform FHT

imaging without having to make significant modifications to the cryomicrotome.

Imaging system 200 may include a housing 202 that houses the various imaging

components of system 200. In some embodiments, housing 202 may include a handle 204 that

allows the user to position housing 202 within chamber 106 of cryomicrotome 100 and/or remove imaging system 200 therefrom, as desired. Housing 202 may be formed of any suitable materials such as plastics, ceramics, or sheet metal and may protect the imaging components of imaging system 200 from the internal climate of chamber 106 of cryomicrotome 100 (e.g., to protect a camera from exposure to the colder temperatures, etc.). At least a portion of housing

202 may also be at least semi-transparent such that light in the white and NIR spectrums may

pass through housing 202. Further, while housing 202 is shown with a primarily cylindrical

shape, other implementations of housing 202 may take on other geometric shapes, e.g., to fit

within the chambers of certain types or models of cryomicrotomes.

In various embodiments, the FHT system may also include one or more support brackets

or other retaining members, to position the imaging components in housing 202 at a suitable

distance within chamber 106 of cryomicrotome 100 relative to tissue specimen 112. For

example, as shown, support mount/bracket 210 may contact the floor of chamber 106 and

support housing 202 at a distance therefrom. While housing 202 may be positioned at any

desired distance from the block face of tissue specimen 112, testing has shown that a distance of

approximately ten inches yields suitable FHT imaging results of the sample while not impinging

the motion of cutting blade 108, as shown in greater detail in FIG. 3.

In some embodiments, bracket 210 may be coupled or otherwise fastened to the floor of

chamber 106 (e.g., via one or more screws, bolts, etc.). In other embodiments, bracket 210 may

be shaped to engage one or more components of cryomicrotome 100 (e.g., to slide under the

structure associated with cutting blade 108, etc.). In addition, bracket 210 may be a separate

component from that of housing 202 (e.g., housing 202 rests on bracket 210), may be fastened or coupled thereto, or may be directly formed as part of housing 202, according to various embodiments.

The imaging components of imaging system 200 may include one or more cameras, such

as one or more charge-coupled device (CCD) camera(s) 206 and one or more illumination light

sources/fibers 208. For example, camera(s) 206 may include a white light camera configured to

capture images within the visible spectrum and/or a fluorescence camera configured to capture

images in the NIR or IR spectrum. Similarly, illumination light sources/fibers 208 may include

one or more fibers to shine fluorescent and/or white light onto the block face of tissue specimen

112 during imaging. Extending out of the back of housing 202 may be cabling 212 that connect

camera(s) 206 and illumination sources/fibers 208 to a computing device 300, as shown in FIG.

4.

In some cases, ambient illumination by room lighting may be sufficiently diffuse such

that imaging system 200 does not require a dedicated white light illumination source. However,

in other embodiments, imaging system 200 may further include one or more white light sources

as part of imaging system 200, such as part of illumination fibers 208 or lights located on the end

of housing 202, or external to housing 202 (e.g., a surgical lamp, a camera flash, etc.).

Camera(s) 206 may be of any suitable type operable to capture images in the white light

and NIR spectrums. For example, one prototype of the FHT system herein uses a high resolution

Canon EOS 700 white light camera available from Canon, Melville, NY, although any other

suitable white light camera can be used in other implementations. For the fluorescence imaging,

suitable systems include the K-FLARE*, and Lab-FLARE@ models R1TM, R1vTM, RP1TM,

RP2TM, RC2TM, FLARE@ (FLuorescence-Assisted Resection and Exploration) imaging systems available from Curadel LLC, Marlboro, MA. Other suitable system components may be used, as desired, without deviating from the teachings herein.

To reduce specular reflections, imaging system 200 may include polarizers with

camera(s) 206 and/or the illumination sources (e.g., illumination fibers 208). For example,

imaging system 200 may use concentric linear polarizers with excitation and emission rotated at

90 degrees, which will reduce specular reflections from the illuminated tissue specimen 112.

FIG. 5 illustrates an example schematic block diagram of computing device 300,

according to various embodiments. As shown, computing device 300 may comprise one or more

interfaces 310 (e.g., wired, wireless, etc.), at least one processor 320, and a memory 340

interconnected by a system bus 350 and powered by a power supply 360.

Interface(s) 310 contain the mechanical, electrical, and signaling circuitry for

communicating data with other computing devices in the FHT system. For example, interfaces

310 may be communicatively coupled to camera(s) 206 and illumination fibers 208 of imaging

system 200 via cabling 212 either directly or via any number of intermediate components. For

example, interface(s) 310 may be in communication with one or more light sources for

illumination fibers 208, to provide control over when the light sources are activated (e.g., to

shine fluorescent light on tissue specimen 112). Further, interface(s) 310 may receive captured

image data from the white light and fluorescence cameras of imaging system 200 for further

image processing.

In some cases, interface(s) 310 may also be in communication with one or more user

interface devices. Generally, a user interface device provides sensory information to a user

and/or receives input from the user via one or more sensors. For example, user interface devices may include, but are not limited to, electronic displays (e.g., to display the resulting images of the tissue block face to the user), pointing devices (e.g., track pads, touch screens, etc.), audio equipment (e.g., speakers, microphones, etc.), and the like. Additionally, interface(s) may also communicatively couple computing device 300 to other computing devices via a hardwired or wireless network (e.g., to convey image data to another device, to receive instructions from another device, etc.).

The memory 340 comprises a plurality of storage locations that are addressable by the

processor 320 and interface(s) 310 for storing software programs and data structures associated

with the embodiments described herein. The processor 320 may comprise hardware elements or

hardware logic adapted to execute the software programs and manipulate the data structures 345,

which may include received sensor data (e.g., captured image data, etc.), operating parameters or

settings, and the like. An operating system 342, portions of which are typically resident in

memory 340 and executed by processor 320, functionally organizes device 300 by, inter alia,

invoking operations in support of software processes and/or services executing on device 300.

These software processes and/or services may comprise, in various embodiments, an

illumination controller process 347 and/or an imaging process 248, as described herein.

It will be apparent to those skilled in the art that other processor and memory types,

including various computer-readable media, may be used to store and execute program

instructions pertaining to the techniques described herein. Also, while the description illustrates

various processes, it is expressly contemplated that various processes may be embodied as

modules configured to operate in accordance with the techniques herein (e.g., according to the

functionality of a similar process). Further, while the processes have been shown separately, those skilled in the art will appreciate that processes may be routines or modules within other processes.

In general, illumination controller 347 may be configured to control when the fluorescent

light source, and possibly the white light source, is activated. As would be appreciated, the light

source itself, such as a light emitting diode (LED), laser, etc., may be in communication with

computing device 300 and may be optically coupled to illumination fibers 208, to emit the

corresponding light onto the block face of tissue specimen 112. For example, illumination

controller 347 may control when imaging system 200 illuminates tissue specimen 112 with NIR

or IR wavelengths, to provoke an excitation response from the fluorophore(s) present within the

tissue.

Imaging process 248 may be operable to acquire and/or process images captured by

imaging system 200. For example, imaging process 248 may send control signals to camera(s)

206 to capture white light and/or fluorescence images of the block face of tissue specimen 112.

In turn, imaging process 248 may receive the captured image data from camera(s) 206 and

perform image processing, as described below, to generate a finalized image for output to an

electronic display. In some embodiments, imaging process 348 may be further configured to

control one or more automated functions of cryomicrotome 100, such as automated actuation of

cutting blade 108, movement of specimen retainer 110, etc.

Referring now to FIG. 6, an example simplified procedure 600 is shown for preparing a

tissue specimen for FHT image acquisition, according to various embodiments. As shown,

procedure 600 may start at step 605 and continue on to step 610 at which the subject

sample/specimen is prepared. In various embodiments, this step may entail selecting a suitable fluorophore for the tissue specimen and injecting the selected fluorophore(s) into the specimen.

For example, as shown previously in Table 1, different fluorophores may have different spectral

properties and applications. After a suitable accumulation time subsequent to the fluorophore

injection, the soft tissue for analysis may be excised.

At step 615, after preparation of the subject specimen to undergo FHT imaging, the

subject may be embedded in a block of OCT compound. Any suitable OCT compound may be

selected for this step. The OCT-encased subject tissue block may then be installed into position

within the cryomicrotome (e.g., as shown in FIG. 1B).

Procedure 600 may also include a step 620 in which the FHT imaging system

components described above are positioned and pointed at the block face of the subject block.

For example, imaging system 200 may be positioned in front of the specimen block face of tissue

specimen 112 within chamber 106 of cryomicrotome 100 for imaging of the block face, as

described above. Notably, the fluorescence components may be positioned such that as much of

the subject specimen as possible is in focus for the camera and the subject subtends the largest

possible field of view without occlusion.

Procedure 600 may also include a step 625 at which a fluorescence channel is selected for

the imaging system. Notably, different NIR channels may be selected, based on the fluorophore

used on the tissue specimen in step 610. If multiple fluorophores are used on the specimen, the

corresponding NIR channels may be selected to overlap the spectral properties of the

fluorophores.

Similar to step 620, procedure 600 may include a step 630 at which a high resolution

white light camera is pointed at the tissue specimen. If, for example, the white light camera and fluorescence camera are both located within the same housing (e.g., housing 202), steps 620 and

630 may be performed at the same time by positioning the housing within the chamber of the

cryomicrotome relative to the tissue specimen.

At step 635 of procedure 600, the user may adjust the positions of the fluorescence and

white light cameras, as needed. For example, based on test images acquired by the cameras, the

positions of the cameras may be further adjusted to ensure that the desired area of the block face

of the tissue is captured, the cameras are in focus, or for any other reason.

In step 640 of procedure 600, once the white light and fluorescence imaging systems are

positioned at a desirable location relative to the block face of the OCT block, the components

may be locked into position. For example, if the imaging components are housed within a single

housing, the position of the housing within the chamber of the cryomicrotome may be solidified,

once the desired position is achieved.

At step 645 of procedure 600, after preparing and mounting the tissue specimen/sample,

the blade(s) of the cryomicrotome may be actuated to shave the OCT block until the tissue is

nearly exposed. In other words, on completion of step 645, the exposed block face may

comprise only a very fine layer of OCT compound in front of the encased tissue for imaging.

Procedure 600 then ends at step 650.

Referring now to FIG. 7, an example simplified procedure 700 for performing FHT on a

tissue specimen is shown, according to various embodiments. In some embodiments, procedure

700 may be performed in whole, or in part, by operating a FHT system having a computing

device (e.g., device 300) in communication with an imaging system (e.g., imaging system 200).

Procedure 700 may start at step 705 and continue on to step 710 where, as described in greater detail above, the room lights may be activated in the room in which the FHT system is located.

Depending on the capabilities of the imaging system, ambient light from the room lights may

provide sufficient white light for purposes of imaging.

At step 715 of procedure 700, the FHT system may acquire an image of the block face of

the tissue specimen using its white light camera. In particular, the computing device of the FHT

system may signal the white light camera to capture a high definition, white light image of the

block face of the tissue specimen within the chamber of the cryomicrotome.

At step 720 of procedure 700, the FHT system may also capture one or more images

using its fluorescence imaging components simultaneously with the step 715 or within a short

time before or thereafter. In some embodiments, the fluorescence imaging components may

capture both white light and fluorescence/NIR images of the block face of the tissue specimen.

For example, the fluorescence camera may capture images of the block face with the room lights

activated, with and without fluorescence illumination, as well.

At step 725 of procedure 700, the room lights may be disabled to remove the white light

source from the tissue specimen. Alternatively, if a dedicated white light source is used, steps

710 and 725 may entail turning the white light source on and off, as needed.

At step 730 of procedure 700, the FHT system may also capture an image of the block

face under fluorescence illumination with the room lights deactivated. Thus, as a result of steps

715, 720, and 730, the FHT system may have any or all of the following distinct images of the

block face: 1.) a white light image captured by the white light camera while the block face was

illuminated with white light (e.g., with the room lights on), 2.) a fluorescence image captured by

the fluorescence imaging system while the block face was illuminated solely with white light, 3.) a fluorescence image captured by the fluorescence imaging system while the room lights and fluorescent light sources were both on, 4.) a fluorescence image captured by the fluorescence imaging system while all light sources were off, and 5.) a fluorescence image captured by the fluorescence imaging system while the white light source was off and the fluorescent lighting source was turned on.

At step 735, the room lights may be reactivated, after completing the imaging of the

block face. In turn, at step 740, the blade(s) of the cryomicrotome may be activated to cut the

OCT block, thereby exposing another portion of the sample for imaging. At or around this time,

any breaks in the protocol defined by the steps above may be noted at a step 745. For example,

if a superfluous image was taken, a note may be may and associated with any of the images

captured of the exposed block face, thereby allowing these images to be discarded or otherwise

ignored.

At step 750 of procedure 700, a decision may be made as to whether the removed layer of

tissue from the OCT block is the last layer of interest. If not, procedure 700 may return to step

715, thereby repeating steps 715-745 for the newly exposed layer. However, if the final layer of

tissue has been imaged, procedure 700 may continue on to step 755 where procedure 700 then

ends.

It should be noted that while certain steps within procedures 600-700 may be optional as

described above, the steps shown in FIGS. 6-7 are merely examples for illustration, and certain

other steps may be included or excluded as desired. Further, while a particular order of the steps

is shown, this ordering is merely illustrative, and any suitable arrangement of the steps may be

utilized without departing from the scope of the embodiments herein. Moreover, while procedures 600-700 are described separately, certain steps from each procedure may be incorporated into each other procedure, and the procedures are not meant to be mutually exclusive.

Referring now to FIG. 8, an illustration 800 is shown of the registration of images within

a FHT system, according to various embodiments. In particular, the FHT computing device

(e.g., device 300 executing imaging process 248) may co-register the various captured images

(e.g., images captured via procedure 700 shown in FIG. 7), to generate the finalized image(s).

The finalized images may then be provided to an electronic display or another user interface

device, for review by a human user.

For purposes of illustration, assume the following labels are assigned to their

corresponding images:

WL FLARE ON: the image 805 captured in the white light channel of the fluorescence

imaging system of the block face illuminated with white light.

WL FLARE OFF: the image captured in the white light channel of the fluorescence

imaging system of the block face with all white lights off but fluorescence excitation light on.

FL FLARE ON: the image 815 captured in the fluorescence channel of the fluorescence

imaging system with the block face illuminated with both fluorescence excitation and white

light.

FL FLARE OFF: the image 820 captured in the fluorescence channel of the

fluorescence imaging system with the block face illuminated solely with fluorescent excitation

light.

WL HIGH ON: the image(s) 810 captured by the high resolution white light camera

with the block face illuminated with white light.

In various embodiments, the computing device may perform image processing by

performing any or all of the following, as shown in FIG. 8:

1. Co-register WLHIGHON images to themselves, creating an aligned image stack.

2. Co-register WLFLAREON to WLHIGHON.

a. FLFLAREON is natively co-registered to WLFLAREON.

3. Co-register WLFLAREOFF to FLFLAREOFF.

4. Perform next-image fluorescence processing on the aligned FLFLAREOFF.

Notably, the computing device may perform co-registration on a per-image basis, with the

exception of the WLHIGHON images.

As would be appreciated, the above procedures are exemplary only and are not intended

to limit the teachings herein. Notably, the above procedures may be of particular use in cases

where the output of the fluorescent light source is comparable to the white light source (e.g.,

stray room lighting, etc.). However, if the fluorescent light signal is sufficiently high, the above

techniques may be modified to allow for single-slice white light and fluorescence images to be

captured simultaneously with the room lights on and natively co-registered to each other. In

particular, optical filters may be used on the fluorescence image in such cases, thereby

simplifying the image captures under different lighting conditions. In some embodiments, the

imaging components may instead comprise the LAB-FLARE@ imaging system from Curadel,

LLC or a similar system. Such systems may allow for the simultaneous acquisition of different images (e.g., color and NIR, etc.), thereby eliminating the need to switch off and on the white light source (e.g., the room lights).

Example - Twine Imaging!

FIGS. 9A-91 illustrate test results of FHT imaging of a fluorophore-infused piece of

twine, according to various embodiments. In particular, imaging of a piece of twine comprising

a plurality of individual strands was performed as a proof-of-concept using a prototype FHT

system employing the techniques herein. During testing, the following steps were performed:

• Kitchen twine was soaked in fluorophore (AlexaFluor 647, 100nM concentration) for 10

minutes.

) The twine was dried and wrapped around a OCT pillow.

• The wrapped pillow was embedded in a larger OCT block and frozen for slicing by the

FHT system.

• Image data was acquired at 50 um thick sections through the OCT block.

• The camera sub-system was positioned approximately 15 cm above the block, resulting

in a field of view (FOV) of ~5 x 5 cm and a transverse pixel size of-0.085 mm.

FIGS. 9A-9B illustrate the original captured images 900-910 of the twine using the FHT

system. As shown, images 900-910 demonstrate a contrast between the portions of the twine

that have high concentrations of the fluorophore and the portions of the twine that do not.

According to various embodiments, the FHT system may further employ subtraction

based deblurring, to produce an image for display. FIGS. 9C-9D illustrate images 900-910,

respectively, after performing subtraction-based deblurring. According to some embodiments, the deblurring may also involve running a Monte Carlo simulation, using a point spread function method, or performing a deconvolution method. The deconvolution method may include one of a measured point spread function kernel, a simulated point spread function kernel, a Richardson

Lucy method, a Weiner filter deconvolution, a Van Cittert deconvolution, a blind deconvolution,

or a regularized deconvolution method.

FIGS. 9E-9F illustrate further images 930-940 of the twine in both original and deblurred

forms, respectively. FIGS. 9G-91 also depict images 950-970 of area 942 of image 940, to

illustrate the application of different image processing techniques to the images. In particular,

image 950 illustrates area 942 in its original form from the fluorescence imaging system. Image

960 in FIG. 9H then illustrates image 950 after performing subtraction-based deblurring.

Finally, in some embodiments, the FHT system may further apply edge-preserving smoothing to

image 960, resulting in image 970 shown in FIG. 91.

Example - Brain Tissue Imaging!

Ex vivo imaging methodologies such as immunohistochemistry, fluorescence imaging,

and autoradiography have been used to study anatomy, physiology and drug or tracer distribution

in either whole bodies or excised organs. These methodologies can follow and accompany in

vivo imaging studies or serve as stand-alone studies themselves. Because ex vivo processing is

relatively expensive and time consuming, often sections and/or images are taken at large

intervals (0.1-1 mm) throughout the entire specimen. Information is lost in these gaps where

sections are not collected or imaged. Further, if a three dimensional model of the specimen is

required, interpolation of largely spaced sections is required and the model may suffer.

To address some of these potential drawbacks, as described above, high resolution white

light and multispectral (700 and 800 nm) fluorescence images may be captured off the block

after every pass of the micro- or macrotome blade using an intraoperative fluorescence imaging

system, thus vastly improving the amount of information captured throughout the specimen and

decreasing total acquisition time. Generally, the process of fluorescent and high resolution white

light data collection and subsequent co-registration and/or three-dimensional reconstruction, is

referred to herein as cryofluorescence tomography or FHT.

Using the FHT techniques herein, organs or small animal whole bodies may be sectioned

(e.g., at 25 microns, etc.) and all images may be captured and acquired in less than 2 hours,

improving some shortcomings of various ex vivo techniques, while enabling the creation of high

resolution 3D models.

To investigate the ability of FHT to study the physiology of the brain, XenoLight

RediJect 2-Deoxy-D-glucose (2-DG, PerkinElmer, 750 nm excitation), a fluorescent glucose

metabolism tracer, was injected into the intrathecal space of a rat. After 1.5 hours of

distribution, the animal was sacrificed, the brain tissue excised, and the sample was

cryopresevered in OCT. With each pass of the microtome blade (25 micron spacing), a high

resolution white light and fluorescent image was acquired.

Further, to study the anatomy of ventricles and subarachnoid space, a fluorescent

zwitterionic compound ZW800-1 (Curadel, LLC) was injected into the rat intrathecal space.

This compound is not expected to cross into the brain parenchyma due to its chemical properties.

As shown, ZW800-1 signal is constrained in the ventricles and subarachnoid space, while it is noticeably absent in brain parenchyma. One can begin to build a high resolution, three dimensional map of the ventricles and subarachnoid space.

Various images captured during testing are shown in FIGS. 1OA-121. Notably, FIG.

10A-10B depict images 1000 and 1010 of the brain tissue with the white light captures colored

gray and the fluorescence images colored orange/purple. FIG. 11A illustrates FHT images 1100

using maximum intensity projection (MIP). FIGS. 111B-111D illustrate sagittal, coronal, and

transverse dual camera static images 1110-1130, respectively (e.g., images 1110-1130 are

combined images of the white light and fluorescence captures). FIG. 11E-1iF illustrate dual

camera, MIP images 1140-1150, respectively.

FIGS. 12A-12C illustrate white light images 1200-1220 of the coronal, axial, and saggital

planes, respectively. FIGS. 12D-12F illustrate fluorescent images 1230-1250 of the 2-DG taken

up in the brain parenchyma along the coronal, axial, and saggital planes respectively. Finally,

images 1260-1280 depict the fluorophore, ZW800-1 constrained in the ventricles and

subarachnoid space.

Thus, as would be appreciated, FHT serves as a multispectral, high resolution, and time

efficient ex vivo tool to study anatomy, physiology, and drug/tracer distribution, either in concert

with in vivo studies or as a stand-alone study.

Example - Pharmacokinetic and Pharmacodynamic Imaging! of Intrathecally

Administered Anti-Sense Oligonucleotides

Antisense oligonucleotides (ASOs) are promising drugs for treating central nervous

system (CNS) disorders due to their specific targeting and extended pharmacological effect. The

development of therapeutics for CNS disorders has been impeded by the inability of most drug molecules to cross the blood brain barrier (BBB) and engage their targets. The intrathecal (IT) dosing route offers a solution for bypassing the BBB and delivering drugs directly to the CNS.

However, determining the pharmacokinetics (PK) and pharmacodynamics (PD) presents unique

challenges imposed by anatomical and functional properties of the IT space and reliance upon ex

vivo histological molecular techniques.

In some aspects, imaging approaches are disclosed herein using radio and fluorophore

labeled ASOs tracking PK. Further aspects of the techniques herein employ neuroreceptor

targeting ASOs to enable tracking of PD using receptor-targeting radiotracers. During testing,

these PK/PD principles were evaluated using two ASOs which target the MALAT1 house

keeping gene and the GABA-A receptor subunit GABRAL. Dynamic SPECT/CT imaging with

the 1251-MALAT1 ASO showed widespread time and dose dependent exposure of the neuroaxis

tissues following lumbar IT injections, with increased exposure in cortical structures versus basal

ganglia. A dosing study using either unlabeled GABRA1 or MALAT1 ASO (n=4 per cohort)

demonstrated progressive decline in 18F-flumazenil uptake specific to the GABRA1 ASO, with

the effect being much greater in cortical structures as compared to basal ganglia. We confirmed

that the reduction of 18F-flumazenil uptake corresponded to GABRA1 mRNA and protein

reduction produced by the ASO.

According to various embodiments, a 3D-FHT imaging technique was developed to

demonstrate the correlation between the distributions of the IT administered Cy7-labeled

GABRA1 ASO with the regional receptor knockdown demonstrated in the 18F-flumazenil. This

3D cryofluorescence imaging technique offers a bridge between in vivo molecular imaging and

ex vivo histology enabling the 3D visualization of the PK/PD relationship for ASO therapy.

Specifically, during testing, two groups of four rats were treated with a single dose of

anti-sense oligonucleotide (ASO) targeting MALAT1 or GABRA A. The rats underwent 1 hour

dynamic 18F-flumazenil PET scans at baseline (day prior to ASO treatment), then at 1, 2, 3, 4

weeks post treatment. Phamacodynamics of treatment is followed by 18F-flumazenil PET,

pharmacokinetics and distribution demonstrated by Cy7-labeled GABRA1 ASO.

Image 1300 in FIG. 13A illustrates averaged MIP, sagittal, coronal and transverse 18F

flumazenil PET area under the curve (AUC) images for MALAT1 and GABRA A ASO-treated

groups at 4 weeks post single-dose treatment. Images were co-registered to a common atlas

space and scaled to units of decay-corrected uCi-min. Note the decreased 18F-flumazenil uptake

post GABRA A versus MALAT1 ASO treatment.

FIG. 13B shows an image 1310 illustrating the MIP, sagittal, coronal and transverse 18F

flumazenil PET (AUC) difference images between MALAT1 and GABRA A ASO treated

groups at 4 weeks post single-dose treatment for N=4 rats per group. Note most voxels show

positive or no change between groups.

FIG. 13C illustrates a graph 1320 of the 18F-flumazenil PET (AUC axis 1324) for volume

of interest on cerebral cortex at baseline and at 1, 2, 3 and 4 weeks post single dose ASO

treatment (time axis 1322). The cortex uptake showed significant reduction in GABRA A versus

MALAT1 targeted treatment a 2, 3, and 4 weeks.

FIG. 13D depicts an image 1330 of the MIP sagittal, coronal and transverse 3D-FHT

images of IT administered Cy7-labeled GABRA1 ASO at 1 hour post administration.

FIG. 13E shows an image 1340 of the MIP sagittal, coronal and transverse 3D-FHT

images of IT administered Cy7-labeled GABRA1 ASO at 4 days post administration.

Clearing! Tissue Slices

As noted during implementation of the techniques herein, the cut tissue slice may "stick"

to the remaining tissue block after slicing, either due to electrostatic, hydrophobic, or other

interactions between the slice and the block. Accordingly, in some embodiments, the FHT

system may further include a mechanism that blows a puff of gas, such as air, or a non-humidity

containing gas such as nitrogen, onto the block face at the end of a slicing cycle to remove any

tissue that might otherwise stick to the block and obscure imaging.

Referring now to FIGS. 14A-14C, examples are shown of a mechanism to remove stuck

tissues slices from an FHT system, according to various embodiments. As shown, the

mechanism may generally include a gas cylinder 1402 that stores a gas such as air, nitrogen, or

argon. As would be appreciated, any form of gas may be selected as desired, depending on the

type of tissue specimen, environmental conditions, etc. For example, in the case of cryo

applications (e.g., in a cryomicrotome), it is important that the gas does not have water vapor

(i.e., humidity), as water vapor can freeze and block the flow the gas. Thus, for such cryo

applications, gasses such as nitrogen and argon may be preferable.

Generally, gas cylinder 1402 may be pneumatically coupled to the FHT system via a

tubing system 1404 and a control mechanism 1406, as shown in FIG. 14A. In particular, control

mechanism 1406 may be configured to regulate the flow of gas from gas cylinder 1402 through

tubing system 1404 and onto the face of the tissue block, to dislodge stuck tissue slices. In other

words, control mechanism may adjust the force of the gas puff so that the gas will reliably

dislodge the tissue slice completely from the block. Similarly, tubing system 1404 may convey

the gas from gas cylinder 1402 onto the tissue block face via control mechanism 1406.

As shown in FIG. 14B, control mechanism 1406 may include a pressure regulator 1408, a

solenoid valve 1410, and control electronics 1412, in various embodiments. Generally, pressure

regulator 1408 controls the amount of gas pressure in tubing system 1404, allowing the user to

adjust the pressure such that any stuck tissue slices are dislodged by the gas exiting the nozzle of

tubing system 1404. Solenoid valve 1410 may also be coupled to tubing system 1404 and

control the flow of gas through tubing system 1404. For example, when actuated, solenoid valve

1410 may block or unblock the flow of gas through tubing system 1404 so as to provide a puff of

gas onto the block face of the tissue specimen. In some embodiments, as shown, control

electronics 1412 may provide electronic or computerized control over solenoid valve 1410

and/or pressure regulator 1408, to control when the system supplies a puff of gas and/or the

pressure of the supplied gas. Such electronics 1412 may either fully automate the actuation of

the system or may allow a user to manually trigger the puff of gas.

FIG. 14C shows tubing system 1404 in greater detail, according to various embodiments.

As shown, tubing system 1404 may include tubing 1414 that couples a nozzle 1416 to gas

cylinder 1402 via control mechanism 1406. Generally, nozzle 1416 may be located at a suitable

distance from the block face of the tissue specimen and may, in some embodiments, be coupled

to the slicing apparatus (e.g., a microtome, etc.), to ensure that nozzle 1416 remains pointed

towards the tissue specimen after each puff of gas. For example, in the case of a cryomicrotome,

tubing 1414 and nozzle 1416 may be located within the temperature-controlled chamber of the

cryostat, thereby cooling the gas to the temperature of the frozen ice block. This prevents

melting of the tissue block that would occur if the gas were at room temperature.

Fiducial Placement

Precise positioning of fiducial markers in a tissue block can be a difficult and time

consuming procedure. Fiducial markers are critical for FHT applications because they permit

orientation of each slice relative to the original block, and permit correction for geometric and

color aberrations during slicing and imaging. In some embodiments, they also permit calculation

of the point-spread function, which is required to correct for the attenuation of light due to

absorption and scattering, and thus reconstruction of the proper fluorescence image.

Referring now to FIGS. 15A-15B, an example apparatus 1500 for precisely placing

fiducial markers in a tissue block is shown, according to various embodiments. As shown,

apparatus 1500 may be configured to engage a tissue chamber 1504 in which a tissue specimen is

retained. Extending through apparatus 1500 may be any number of apertures through which

fiducials 1502 may be placed, thereby inserting fiducials 1502 into the tissue specimen.

Apparatus 1500 may be formed of any suitable material such as ABS plastic or the like.

Preventing! Damage to the Cutting Blade

Damage to the cutting blade of the histological slicing instrument may also be

problematic in an FHT system, especially with automated and semi-automated systems. This

occurs when the specimen retainer 110, typically made of metal, moves into the path of the

cutting blade.

Referring now to FIG. 16, an example of multi-colored fiducial markers 1502 is shown.

To prevent damage to the blade of the slicing instrument, the fiducial markers 1502, such as

pieces of dry Angel hair pasta, may be dipped in a solution of chemical or paint to mark one end

with a color different from the original color. For example, if fiducial 1502a is gray in color, the

distal 1-2 mm might be colored black. Or, if fiducial 1502b is black, such is the case with squid ink-infused pasta, the distal 1-2 mm might be painted or bleached to a gray or white color.

Then, the fiducial 1502 is placed onto the specimen retainer 110 such that the end closest to the

block support has the alternate color of a specified length ("the unsafe zone").

After the tissue block is placed in the slicer, and slicing begins, custom imaging software

may continuously identify both the position and color of the fiducial 1502. When the color

changes from the primary color to the alternate color, the software will stop all slicing, thus

preventing damage to the blade. In additional embodiments, the colors throughout the vertical

height of the fiducial could be selected so that the exact depth of each slice could be estimated.

For example, if a 5 mm fiducial changed color every mm from red, to orange, to blue, to green,

to yellow, the depth of cutting could be estimated by imaging the transition from one color to

another and interpolating based on a known tissue slice depth.

The techniques herein, therefore, provide for the mounting of a FHT imaging system

within the chamber of a cryomicrotome, to perform FHT imaging on the block face of a tissue

specimen within the chamber. In some aspects, the imaging components of the system may be

located within a transportable housing, thereby protecting the components from the conditions

within the chamber and allowing a user to install, position, and remove the imaging components

from the cryomicrotome as desired. Thus, the FHT system herein can be easily adapted for use

with any number of existing cryomicrotomes without significant modification.

The methods according to the inventive concepts may be embodied as a non-transitory

computer program product. Any combination of one or more computer readable storage

device(s) or computer readable media may be utilized. The computer readable medium may be a

computer readable storage medium. A computer readable storage device may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage device would include the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory

(ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical

fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a

magnetic storage device, or any suitable combination of the foregoing. In the context of this

document, a computer readable storage device may be any tangible device or medium that can

store a program for use by or in connection with an instruction execution system, apparatus, or

device. The term "computer readable storage device," or variations thereof, does not encompass

a signal propagation media such as a copper cable, optical fiber or wireless transmission media.

Program code embodied on a computer readable storage device or computer readable

medium may be transmitted using any appropriate medium, including but not limited to wireless,

wireline, optical fiber cable, RF, etc., or any suitable combination of the foregoing.

Computer program code for carrying out operations for aspects of the present invention

may be written in any combination of one or more programming languages, including an object

oriented programming language such as Java, Smalltalk, C++ or the like and conventional

procedural programming languages, such as the "C" programming language or similar

programming languages. The program code may execute entirely on the user's computer, partly

on the user's computer, as a stand-alone software package, partly on the user's computer and

partly on a remote computer or entirely on the remote computer or server. In the latter scenario,

the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service

Provider).

Aspects of the present invention are described herein with reference to flowchart

illustrations and/or block diagrams of methods, apparatus (systems) and computer program

products according to embodiments of the invention. It will be understood that each block of the

flowchart illustrations and/or block diagrams, and combinations of blocks in the flowchart

illustrations and/or block diagrams, can be implemented by computer program instructions.

These computer program instructions may be provided to one or more processors of one or more

general purpose computers, special purpose computers, or other programmable data processing

apparatuses to produce a machine, such that the instructions, which execute via the one or more

processors of the computers or other programmable data processing apparatuses, create means

for implementing the functions/acts specified in the flowchart and/or block diagram block or

blocks.

These computer program instructions may also be stored in one or more computer

readable storage devices or computer readable media that can direct one or more computers, one

or more other programmable data processing apparatuses, or one or more other devices to

function in a particular manner, such that the instructions stored in the one or more computer

readable storage devices or computer readable medium produce an article of manufacture

including instructions which implement the function/act specified in the flowchart and/or block

diagram block or blocks.

The computer program instructions may also be loaded onto one or more computers, one

or more other programmable data processing apparatuses, or one or more other devices to cause a

series of operational steps to be performed on the one or more computers, one or more other

programmable data processing apparatuses, or one or more other devices to produce a computer

implemented process such that the instructions which execute on the one or more computers, one

or more other programmable data processing apparatuses, or one or more other devices provide

processes for implementing the functions/acts specified in the flowchart and/or block diagram

block or blocks.

The terminology used herein is for the purpose of describing particular embodiments only

and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an"

and "the" are intended to include the plural forms as well, unless the context clearly indicates

otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used

in this specification, specify the presence of stated features, integers, steps, operations, elements,

and/or components, but do not preclude the presence or addition of one or more other features,

integers, steps, operations, elements, components, and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of all means or step plus

function elements in the claims below are intended to include any structure, material, or act for

performing the function in combination with other claimed elements as specifically claimed.

The description above has been presented for purposes of illustration and description, but is not

intended to be exhaustive or limited to embodiments in the form disclosed. Many modifications

and variations will be apparent to those of ordinary skill in the art without departing from the scope

and spirit of the invention. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

Throughout this specification and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (12)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A fluorescence histo-tomography (FHT) system comprising a housing having a handle; one or more cameras located within the housing; a fluorescence excitation light source located within the housing; and a support mount coupled to a slicing apparatus and configured to removably position the housing with respect to a chamber of the slicing apparatus such that the one or more cameras and fluorescence excitation light source are aimed towards a block face of a tissue specimen retained within the chamber.

2. The FHT system as in claim 1, wherein the support mount is removably positionable on a floor of the chamber.

3. The FHT system as in claim 1, further comprising: processing circuitry that includes a processor configured to execute a process and a memory to store the process executed by the processor, the process when executed operable to: control a first camera of the one or more cameras to capture a first image of the block face in a visible spectrum; control a second camera of the one or more cameras to capture a second image of the block face in a near infrared or infrared spectrum; process the first image and the second image to form a combined image; and provide the combined image to an electronic display.

4. The FHT system as in claim 1, further comprising a gas container storing a pressurized gas; a nozzle coupled to the gas container and positionable within the chamber of the slicing apparatus to direct the nozzle towards the tissue specimen; and a control mechanism that controls a flow of the pressurized gas towards the tissue specimen within the chamber via the nozzle.

5. The FHT system as in claim 1, further comprising: one or more multi-colored fiducials for insertion into the tissue specimen.

6. The FHT system as in claim 1, wherein the slicing apparatus comprises a cryomicrotome.

7. The FHT system as in claim 1, further comprising: a fiducial positioning apparatus forming a plurality of apertures through which fiducials may be inserted around the tissue specimen.

8. The FHT system as in claim 1, wherein the one or more cameras are configured to detect a fluorophore within the tissue specimen at a wavelength between a range of approximately 200 nm to 1000 nm, when the block face of the tissue specimen is illuminated with the fluorescence excitation light source.

9. A method of performing fluorescence histo-tomography (FHT) comprising: capturing, by an imaging device mounted with respect to a chamber of a slicing apparatus by a support mount coupled to the slicing apparatus and configured to removably position the imaging device with respect to the chamber, a first image of a first block face of a tissue specimen retained within the chamber; capturing, by the imaging device, a second image of the first block face under fluorescence excitation illumination; and processing at least the first image and the second image to form a combined image.

10. The method as in claim 9, wherein the first image is captured in a visible spectrum, and the second image is captured in an infrared spectrum.

11. The method as in claim 9, wherein the first image is captured under illumination with light in a visible spectrum.

12. The method as in claim 9, further comprising: exposing, using the slicing apparatus, a second block face of the tissue specimen; and generating a second combined image of the second block face.